Counting the number and types of cells in a sample has been and continues to be an important diagnostic tool. For example, determining the number of white blood cells (or leukocytes) in a blood sample can provide an indication of infection. Determining the number of platelets, red blood cells and other hematopoietic system components (including reticulocytes) also can provide the clinician with information on the status of a patient's system. More recently, the increase in incidence of AIDS has made counting a specific population of leukocytes extremely important.
AIDS is a condition which results when an individual has become infected with HIV. Progression of the infection generally renders the individual immunodeficient, and as a result, often leads to death from lethal opportunistic infections such as Pneumocystis carinii pneumonia. The mechanism of HIV infection which results in AIDS is believed to be mediated through the binding of HIV to a subset of T-cells which are identified by the CD4 surface antigen. By infecting and mediating the destruction of this subset of T lymphocytes, the individual infected with HIV loses the ability to respond to opportunistic infections and pathogens.
HIV infections progress through a number of different clinical stages which may be distinguished in a variety of ways. One presently accepted classification system for charting the progress of the disease from initial exposure through the latter stages is described in the Walter Reed Classification System.
A number of criteria go into evaluating each of the several stages. For example, the presence or absence of antibodies to HIV or the presence or absence of the virus itself are used as an indication of initial exposure to HIV (WR1). Subsequently, the number of CD4.sup.+ lymphocytes in the blood may be measured. A decrease in the number of CD4.sup.+ lymphocytes indicates that The disease has progressed (WR3). Accordingly, accurately determining the number of CD4.sup.+ lymphocytes in an AIDS patient is clinically important. (For a further description of the Walter Reed Classification System and the clinical aspects of AIDS, see Redfield et al., Sci. Amer., 259:70 (1988).)
In another example, U.S. Pat. No. 4,677,061 describes the importance of determining the ratio of specific cell types in the monitoring of autoimmune patients, particularly patients with multiple sclerosis. In this patent, the ratio of CD4.sup.+ or CD8.sup.+ cells to subsets thereof bearing cellular differentiation antigens is determined. Particularly useful is the ratio of CD4.sup.+ to Lp220.sup.+ cells.
In each instance, counting the number of cells in a given volume of blood is critical to the use of the information. Standard values for many components of the hematopoietic system are known, and it is the measurement of deviation from that standard that is of clinical significance. Accordingly, there have been developed several methods for counting such cells.
Perhaps the oldest method involves the microscopic examination of a whole blood sample (or some fraction or component thereof). The sample is placed on a slide which has been divided into specific fields, and the clinician counts the cells within each field. The method is dependent upon the skill of the clinician in counting the cells but also in distinguishing between cells types. The latter problem can be addressed by selective staining or tagging of specific cells with a variety of dyes and/or immunofluorescence markers; however, the inaccuracies due to subjectivity in manual counting cannot be avoided.
Automated counting methods have been developed in an attempt to incorporate the benefits provided by selective staining but also to reduce the error attributable to the technician. In such systems, the goal is speed with accuracy. One example of such a system involves the electronic counting of cells in a liquid sample. In this example, a known volume of liquid is sent through an instrument having a pair of electrodes. Cells of different sizes can be distinguished based upon the electrical impedance generated as each cell passes between the electrodes. U.S. Pat. No. 2,656,508 describes one system of this type.
One drawback to that system is that the relative counts of different sized particles can be determined but not the absolute counts in a specific volume. In U.S. Pat. No. 4,110,604, a method is described in which absolute counts of platelets can be determined based upon electrical impedance. The number of red blood cells is counted as is the number of platelets. Then, by knowing or determining the number of red blood cells in a given unit of volume, an equation can be used to arrive at the number of platelets in the same unit volume. Alternatively, a reference particle could be included in the sample at a known concentration, and then the reference particle is counted along with the platelets. By knowing the concentration of reference particles, one can determine the concentration of platelets.
A drawback to this system, however, is that it is most useful in distinguishing between cells based upon their physical characteristics, such as size. Not all cells are capable of discrimination based on size. For example, it is not possible to distinguish between CD4.sup.+ and CD8.sup.+ lymphocytes based upon size. Accordingly, other instruments (e.g., flow cytometers) have been developed that combine both measurements and correlate physical characteristics with fluorescence.
Flow cytometry comprises a well known methodology for identifying and distinguishing between different cell types in a non-homogeneous sample. The sample may be drawn from a variety of sources such as blood, lymph, urine, or may be derived from suspensions of cells from solid tissues such as brain, kidney or liver. In the flow cytometer, cells are passed substantially one at a time through one or more sensing regions where each cell is illuminated by an energy source. The energy source generally comprises means that emits light of a single wavelength in a sensing region such as that provided by a laser (e.g., He/Ne or argon) or a mercury arc lamp with appropriate bandpass filters. Different sensing regions can include energy sources that emit light at different wavelengths.
In series with each sensing region, various light collection means, such as photomultiplier tubes, are used to gather light that is refracted by each cell (generally referred to as forward light scatter), light that is reflected orthogonal to the direction of the flow of the cells through a sensing region (generally referred to as orthogonal light scatter) and one or more light collection means to collect fluorescent light that may be emitted from the cell as it passes through a sensing region and is illuminated by the energy source. Light scatter is generally correlated with the physical characteristics of each cell.
Flow cytometers further comprise data recording and storage means, such as a computer, wherein separate channels record and store the light scattered and fluorescence emitted by each cell as it passes through a sensing region (i.e., the data collected for each cell comprises a "recorded event"). By plotting orthogonal light scatter versus forward light scatter in either real time or by reanalysis of the data after the events have been recorded, one can distinguish between and count, for example, the granulocytes, monocytes and lymphocytes in a population of leukocytes. By gating on only lymphocytes, for example, using light scatter and by the use of appropriate immunofluorescence markers, such as monoclonal antibodies labelled with fluorochromes of different emission wavelength, one can further distinguish between and count cell types within the lymphocyte population (e.g., between CD4.sup.+ and CD8.sup.+ lymphocytes). U.S. Pat. Nos. 4,727,020, 4,704,891 and 4,599,307 describe the arrangement of the various components that comprise a flow cytometer and also the general principles of its use.
While it is possible using the above-described methods to count the number of cells in a sample and to distinguish between various cell populations, the number of cells counted will be relative (i.e., it will not give an absolute count for a specific volume of blood). Generally, these methods require that red blood cells be substantially removed from the sample. One reason is because the light scatter of the red blood cells and leukocytes is substantially overlapping making their differentiation based on light scatter alone difficult. Another reason is that in order to count leukocytes in a more rapid manner the number of red blood cells must be reduced because the number of red blood cells to leukocytes is approximately 1,000 to 1. Accordingly, practitioners in the field routinely lyse whole blood or separate out the blood cell components by density dependent centrifugation.
In addition to the step required for whole blood separation, other steps are routinely involved. For example, once a lysed blood preparation is made, immunofluorescence markers can be added. Unbound antibodies, then, are routinely washed from the cells. After that step, a fixative is added. Finally, cells are run on a flow cytometer. Each step introduces not only the possibility for error, but also the loss of cells from the sample. In addition, each step increases the risk to the technician of being exposed to contaminated blood. Using these traditional flow cytometric methods, therefore, the number of cells in a given volume of blood cannot be easily or accurately determined.
Thus, in each of the presently described systems, there are one or more obstacles that prevent one from accurately determining the absolute count of specific cells in a heterogeneous sample of blood. These obstacles are not overcome by the mere addition of a reference particle, as described in U.S. Pat. No. 4,110,604, with flow cytometry. Several drawbacks remain.
A major drawback to the use of flow cytometers is that unless the fluorescence channels and optical alignment of each flow cytometer is calibrated to read the same, there is no assurance as to the source of variation in a sample. It is likely that one instrument will give different readings on the same sample on different days if it was aligned and/or calibrated differently each day. Similarly, there is no assurance that any two instruments will provide the same results even if properly set up. Accordingly, while flow cytometry provides a better measure of identifying and distinguishing between cells in a sample, its present use as a clinical instrument is diminished by the limitations in set up and operation. What is required is a single system or method that will allow one to accurately count cells in a sample and be assured that the results from one instrument are consistent from sample to sample as well as consistent with results obtained from other instruments.
Another obstacle is to decrease or limit the exposure of the technician to an infectious sample. Traditionally, cell fixatives, such as paraformaldehyde, have been added to flow cytometry samples not only to "fix" the cell/antibody interaction but also to inactivate infectious agents that may be in the sample. Fixation traditionally has been done after staining. As a result, the technician was required to mix the sample with the immunofluorescence marker(s) and then fix. This then required separate containers for each reagent increasing the number of steps needed before a sample can be run, thus raising the possibility for error and, as importantly, the possibility for exposure.
Finally, it has be traditionally required to mix the immunofluorescence marker(s) with a small sample volume. Typically, 20 .mu.l of reagent(s) were added to 50 .mu.l of sample. It was believed that the total volume containing the cells and reagents should be small so that complete staining would occur.
The present invention overcomes all of these obstacles and provides a one step test for absolute counting of one or more specific populations of cells in an unlysed whole blood sample.